DESCRIPTION: (Applicant's Description) The presence of estrogen receptor (ER) is an important prognostic factor for breast cancer survival, critical in predicting the potential for response to endocrine therapy. However, response to endocrine therapies such as tamoxifen and raloxifene require a functional ER. A sizable proportion (thirty percent) of breast cancers appear to be ER positive, yet are not hormone responsive, and many others develop hormone insensitivity and relapse, even when ER is apparently present. This paradox may result from an imprecise determination of receptor status: the receptor protein may be physically present, yet have mutations that inactivate or alter its function or half-life in the cell. Often tumors show an abnormally high level of ER, which is often interpreted by clinicians as simply an increased indication for effective tamoxifen therapy, though the high levels and its consequences remain underexplored. Some reports have implicated increased transcription of the ER gene from alternate promoter elements, though these studies are primarily based in transformed cell lines where gene regulation is notoriously irregular. This research proposes to look at function of ER from breast cancer samples that show ER overexpression, i.e. abnormally high levels of specific ER:estradiol binding that can approach 100 times normal. It consists of two specific aims, using archived frozen tumors with elevated ER levels: 1) to identify receptor mutations in the hormone binding domain affecting ER folding and transcriptional function, and 2) to assay ER DNA binding and half-life, correlating the data with clinical outcome for these patients. Analysis will compare both ER-alpha and ER-beta forms to see if ER-beta expression is a significant component of the elevated estradiol binding. These aims address the potential consequences of elevated ER protein with respect to efficacy of primary therapy using tamoxifen. If ER is transcriptionally normal, these tumors will have overexpressed many estrogen responsive genes, including paracrine growth factors. If ER is mutated, tamoxifen therapy may have been ineffective or even detrimental. This research proposal employs a novel and rapid yeast assay system to detect functional changes in ER.